involvement of collapsin response mediator protein 2 in posttraumatic sprouting in acquired epilepsy

186 Pages·2014·4.28 MB·English

by  Sarah Wilson

INVOLVEMENT OF COLLAPSIN RESPONSE MEDIATOR PROTEIN 2
IN POSTTRAUMATIC SPROUTING IN ACQUIRED EPILEPSY

Sarah Marie Wilson

Submitted to the faculty of the University Graduate School
in partial fulfillment of the requirements
for the degree
Doctor of Philosophy
in the Program of Medical Neuroscience,
Indiana University

May 2014
Accepted by the Graduate Faculty, of Indiana University, in partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

________________________
Gerry S. Oxford, Ph.D., Chair

________________________
Rajesh Khanna, Ph.D.
Doctoral Committee

________________________
Joanna Jen, M.D.,Ph.D.

________________________
Zao C. Xu, M.D., Ph.D.
March 17, 2014

________________________
Xiaoming Jin, Ph.D.

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© 2014
Sarah Marie Wilson

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DEDICATION
This thesis is dedicated to my parents for always pushing me to achieve my goals
and to my husband for his unwavering support.

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ACKNOWLEDGEMENTS
The completion of this thesis and the work described herein would not be possible
without the contribution of so many people. First and foremost I would like to thank my
mentor Dr. Rajesh Khanna for his guidance throughout my graduate school career. His
enthusiasm for science is what drew me to his laboratory and remains something I admire
greatly. Truly, I will consider myself successful if I embrace my career with even half of
his drive and determination. I thank the members of my dissertation committee, Dr.
Gerry Oxford, Dr. Zao Xu, Dr. Xiaoming Jin, and Dr. Joanna Jen for their insight and
ideas, as well as assistance with experimental design and interpretation. I thank the
following IU School of Medicine faculty: Dr. Andy Hudmon, Dr. Ted Cummins, Dr.
Fletcher White, Dr. Grant Nicol, and Dr. Michael Vasko for their willingness to answer
my endless questions. It was a great comfort knowing that your door was always open, at
least figuratively. Additionally I thank Dr. Grant Nicol, Dr. Cynthia Hingtgen, Dr. Ted
Cummins, and Dr. Andy Hudmon for their efforts on behalf of the Medical Neuroscience
Program. I thank Nastassia Belton and Brittany Veal of the Stark Neurosciences
Research Institute office for basically anything and everything, as well as Monica Henry
for ensuring that the transition to graduate school went as smoothly as possible.
Graduate school would be much more daunting if not for the people with whom it
is shared. I would like to thank Matthew Ripsch, Dr. Joel Brittain, and Dr. Nicole
Ashpole for their patience and expertise while teaching me new techniques. I thank past
and current members of the Khanna Laboratory: Dr. Joel Brittain for his taste in music
and for always getting my vague movie references, Dr. Yuying Wang for her hard work,
Weina Ju for her positive attitude and love of bling, Erik Dustrude for his shared sense of
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dry humor, and Xiao-Fang Yang for all the times that she made fun of Erik. I thank
Alicia Garcia, Kisan Shah, and Angelica Dixon for doing all the things that I forgot to do
(dishes, autoclaving the trash, etc). I most certainly am indebted to Jessica Head and
Stephanie Martinez for their efforts in the microscope room. I also thank present and
former members of the laboratory of Dr. Harold Kohn for providing lacosamide
compounds, Dr. May Khanna for her attempts at teaching me the intricacies of protein
chemistry, members of the White Laboratory for allowing me to infiltrate their space and
use their instruments at will, and Dr. Joel Brittain for the use of the 1 million constructs
that he created. I thank the members of the Indiana Spinal Cord and Brain Injury
Research Group for the use of their core facility. I thank the Paul and Carole Stark Pre-
Doctoral Fellowship for financial support of my research. I would be remiss not to thank
the mentors from whom I learned so much during my undergraduate research: Dr. Todd
Mowery and Dr. Preston Garraghty.
Last but certainly not least I would like to thank my fellow graduate students Dr.
Nicole Ashpole, Derrick Johnson, Aarti Chawla, and Yohance Allette-Noel for their
friendship, willingness to listen to my rants, shared love of caffeine, and taste in music
(you know who you are). I thank Jessica Pellman and Valerie Fako for being 2 of the
best friends that I have had and especially for introducing me to the local craft beer.
Most of all I thank my family. My parents have done nothing but encourage me. I am
forever indebted to my husband for putting up with all of the late nights and weekends
spent in lab, just don’t tell him I said that.

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Sarah Marie Wilson
INVOLVEMENT OF COLLAPSIN RESPONSE MEDIATOR PROTEIN 2 IN
POSTTRAUMATIC SPROUTING IN ACQUIRED EPILEPSY

Posttraumatic epilepsy, the development of temporal lobe epilepsy (TLE)
following traumatic brain injury, accounts for 20% of symptomatic epilepsy.
Reorganization of mossy fibers within the hippocampus is a common pathological
finding of TLE. Normal mossy fibers project into the CA3 region of the hippocampus
where they form synapses with pyramidal cells. During TLE, mossy fibers are observed
to innervate the inner molecular layer where they synapse onto the dendrites of other
dentate granule cells, leading to the formation of recurrent excitatory circuits. To date,
the molecular mechanisms contributing to mossy fiber sprouting are relatively unknown.
Recent focus has centered on the involvement of tropomycin-related kinase
receptor B (TrkB), which culminates in glycogen synthase kinase 3β (GSK3β)
inactivation. As the neurite outgrowth promoting collapsin response mediator protein 2
(CRMP2) is rendered inactive by GSK3β phosphorylation, events leading to inactivation
of GSK3β should therefore increase CRMP2 activity. To determine the involvement of
CRMP2 in mossy fiber sprouting, I developed a novel tool ((S)-LCM) for selectively
targeting the ability of CRMP2 to enhance tubulin polymerization. Using (S)-LCM, it
was demonstrated that increased neurite outgrowth following GSK3β inactivation is
CRMP2 dependent. Importantly, TBI led to a decrease in GSK3β-phosphorylated
CRMP2 within 24 hours which was secondary to the inactivation of GSK3β. The loss of
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GSK3β-phosphorylated CRMP2 was maintained even at 4 weeks post-injury, despite the
transience of GSK3β-inactivation.
Based on previous work, it was hypothesized that activity-dependent mechanisms
may be responsible for the sustained loss of CRMP2 phosphorylation. Activity-
dependent regulation of GSK3β-phosphorylated CRMP2 levels was observed that was
attributed to a loss of priming by cyclin dependent kinase 5 (CDK5), which is required
for subsequent phosphorylation by GSK3β. It was confirmed that the loss of GSK3β-
phosphorylated CRMP2 at 4 weeks post-injury was likely due to decreased
phosphorylation by CDK5. As TBI resulted in a sustained increase in CRMP2 activity, I
attempted to prevent mossy fiber sprouting by targeting CRMP2 in vivo following TBI.
While (S)-LCM treatment dramatically reduced mossy fiber sprouting following TBI, it
did not differ significantly from vehicle-treated animals. Therefore, the necessity of
CRMP2 in mossy fiber sprouting following TBI remains unknown.

Gerry S. Oxford, Ph.D.

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TABLE OF CONTENTS
LIST OF TABLES ........................................................................................................... xiv
LIST OF FIGURES ...........................................................................................................xv
FIGURE CONTRIBUTIONS ......................................................................................... xvii
LIST OF ABBREVIATIONS ........................................................................................ xviii
CHAPTER 1. INTRODUCTION ........................................................................................1
1.1. Temporal Lobe Epilepsy: A progressive, acquired phenomenon ..............................2
Background .....................................................................................................................2
Animal models ................................................................................................................5
1.2. Mossy Fiber Sprouting ...............................................................................................7
Background .....................................................................................................................7
Lesion-induced sprouting ................................................................................................8
Mossy fiber sprouting in TLE .......................................................................................12
Activity dependence ......................................................................................................14
Functional consequences ...............................................................................................15
1.3. Mechanisms of Mossy Fiber Sprouting ....................................................................20
Background ...................................................................................................................20
BDNF ............................................................................................................................23
GSK3β ...........................................................................................................................26
1.4. Collapsin Response Mediator Protein 2 (CRMP2) ..................................................27
Background ...................................................................................................................27
Post-translational regulation .........................................................................................30
Potential involvement in TLE .......................................................................................31
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1.5. (R)-Lacosamide ........................................................................................................32
Background ...................................................................................................................32
Mechanism of action .....................................................................................................33
Controversy ...................................................................................................................33
Impact on epileptogenesis .............................................................................................36
1.6. Thesis Aims ..............................................................................................................41
CHAPTER 2. MATERIALS AND METHODS ...............................................................43
2.1. Lacosamide compounds and recombinant proteins .................................................44
2.2. NT-647 labeling of CRMP2 proteins .......................................................................44
2.3. Microscale Thermophoresis (MST) binding analysis ..............................................47
2.4. Primary cortical neuron culture ................................................................................47
2.5. Transfection of cortical neuron cultures ...................................................................48
2.6. Immunocytochemistry ..............................................................................................48
2.7. Sholl analysis ............................................................................................................50
2.8. siRNA knockdown of CRMP2 .................................................................................50
2.9. Co-Immunoprecipitation ..........................................................................................54
2.10.. Immunoblot assay ..................................................................................................54
2.11. Turbidimetric assay for tubulin polymerization .....................................................55
2.12. Synaptic bouton size ...............................................................................................55
2.13. Glutamate release ..................................................................................................57
2.14. ImageXpress neurite nutgrowth .............................................................................58
2.15. Whole-cell patch-clamp recordings ........................................................................59
2.16. Oxygen Glucose Deprivation (OGD) .....................................................................59
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